RC586 have <97.7% sequence identity with the rpoB sequences of all V. cholerae and V. mimicus strains included in this study. In a comparative DNA-DNA hybridization and ANI analysis, Adékambi et al. [23] demonstrated that rpoB <97.7% correlated with DNA-DNA hybridization <70% and ANI <95%, both being interpreted as demarcation thresholds for bacteria. All V. cholerae strains included in this study showed >99.5% rpoB sequence similarity with V. cholerae N16961 (data not shown). Based on a standard MLSA for the Vibrionaceae [21], Vibrio sp. RC341 and Vibrio sp. RC586 both have <95% pair-wise

similarity with V. cholerae, V. mimicus, V. vulnificus, and V. parahaemolyticus strains. All V. cholerae strains and both V. mimicus strains Lenvatinib used in this analysis demonstrated >95% similarity between concatenated genes of like-species (data not shown). Karlin’s dissimilarity signatures were also calculated between these two genomes and the Vibrio genomes used in this study. Vibrio sp. RC586 shared >10 dissimilarity with all V. cholerae (11.5 to 16.2), V. vulnificus (19.6), and V. parahaemolyticus (41.6) genomes, and > 7 with both V. mimicus strains. Vibrio sp. RC341 shared >10 dissimilarity for all V. cholerae (10.2 to 14) except V. cholerae B33 (9.4) and TMA21 (9.8). Vibrio sp. RC341 shared >10 genome signature dissimilarity with V. parahaemolyticus find more (40.2), V. vulnificus (16.3), and both V. mimicus (>14) genomes. Vibrio

sp RC341 and RC586 learn more shared a genomic dissimilarity of 8.7 with each other. Taken together these data indicate that Vibrio sp. RC341 and Vibrio sp. RC586 are new species with a high genomic relatedness to V. cholerae and V. mimicus. Evolution of Vibrio sp. RC341 and Vibrio sp. RC586 Lineages The phylogenies of Vibrio sp. RC341 and Vibrio sp. RC586 were inferred by constructing a supertree, using a 362,424 bp homologous alignment of V. cholerae, V. mimicus, and the new species (Figure 2). Based on the supertree analysis Vibrio sp. RC341 and Vibrio sp. RC586 are deeply rooted in SIS3 mw ancestral nodes, suggesting ancient evolution of the

two species. Results of this phylogenetic analysis suggest the Vibrio sp. RC341 lineage evolved from a progenitor of the V. cholerae and V. mimicus lineages (Figure 2), a finding supported by strong bootstrap support and further evidenced by the evolutionary distance of V. cholerae and V. mimicus from Vibrio sp. RC341 (see Additional file 7). The two V. mimicus strains are interspersed among V. cholerae, with respect to evolutionary distance, suggesting that evolutionary distances of V. cholerae and V. mimicus are equidistant from Vibrio sp. RC341 (see Additional file 7). Figure 2 Neighbor-joining tree based on 362,424 bp alignment of homologous sequences using the Kimura-2 parameter for nucleotide substitution. The bootstrap supports, as percentage, are indicated at the branching points. Bar represents 0.005 substitutions per site. The phylogeny of Vibrio sp.